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Summary
Researchers explored how DNA supercoiling affects transcription. Supertwisting DNA with gyrase slightly inhibited bacterial RNA polymerase but greatly stimulated wheat germ RNA polymerase II activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Nuclear DNA from HeLa cells can be isolated unbroken and supercoiled.
- DNA supercoiling is a critical factor in DNA replication and transcription.
Purpose of the Study:
- To investigate the impact of DNA supercoiling on transcription by different RNA polymerases.
- To compare the effects of supercoiling on bacterial and eukaryotic RNA polymerases.
Main Methods:
- Isolation of unbroken, supercoiled nuclear DNA from HeLa cells.
- Preparation of an allomorphic series of DNA templates (nuclear and PM2 viral DNA) using DNA gyrase and untwisting enzyme.
- In vitro transcription assays using wheat germ RNA polymerase II and Escherichia coli RNA polymerase.
Main Results:
- Relaxed DNA templates were transcribed slowly by both RNA polymerases.
- Supertwisting DNA with gyrase slightly inhibited transcription by E. coli RNA polymerase.
- Supertwisting DNA with gyrase dramatically stimulated transcription by wheat germ RNA polymerase II.
Conclusions:
- DNA supercoiling differentially affects transcription by bacterial and eukaryotic RNA polymerases.
- Wheat germ RNA polymerase II activity is highly sensitive to changes in DNA superhelicity.